Yoshimoto Shogo, Ohara Yuki, Nakatani Hajime, Hori Katsutoshi
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.
Microb Cell Fact. 2017 Jul 18;16(1):123. doi: 10.1186/s12934-017-0740-7.
Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be increased, stabilizes enzymatic activity, and permits repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner part of a gel, gel fragility, cell leakage from the support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5. This approach is expected to solve the drawbacks of conventional immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials.
We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is drastically diminished at ionic strengths lower than 10 mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam in a salt solution were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness in a salt solution and could rapidly be re-immobilized. The cells expressing the ataA gene maintained their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake in a flask. We also demonstrated that both bacterial cells and a support used in a reaction could be reused for a different type of reaction after detachment of the initially immobilized cells from the support and a subsequent immobilization step.
We invented a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation involving a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the efficiency and productivity of microbial production processes.
微生物细胞固定化是有效利用全细胞催化剂的一项重要策略，因为它简化了产物分离，能够提高细胞浓度，稳定酶活性，并允许重复或连续使用生物催化剂。然而，传统的固定化方法存在实际局限性，如凝胶内部传质受限、凝胶易碎、细胞从载体基质中泄漏以及对细胞活力和催化活性产生不利影响。我们之前展示了一种利用AtaA固定细菌细胞的新方法，AtaA是不动杆菌属Tol 5中发现的三聚体自转运粘附素家族的一员。这种方法有望解决传统固定化方法的缺点。然而，与所有其他固定化方法一样，使用载体材料会增加生物过程的成本以及后续的废料。
我们发现，从Tol 5细胞中分离出的AtaA分子在离子强度低于10 mM时粘性急剧降低，且在去离子水中无法粘附，这也抑制了由AtaA介导的细胞粘附。分别通过冲洗和摇晃，固定在盐溶液中的微孔板和聚氨酯泡沫上的细胞在去离子水中会脱落。脱落的细胞在盐溶液中恢复其粘附性，并可迅速重新固定。表达ataA基因的细胞在四个重复的固定化和脱附循环中均保持其粘附性，并且通过在烧瓶中摇晃10分钟可反复固定到聚氨酯泡沫上。我们还证明，在将最初固定的细胞从载体上脱附并进行后续固定化步骤后，反应中使用的细菌细胞和载体均可用于不同类型的反应。
我们基于AtaA分子的盐依赖性粘附发明了一种独特的可逆固定化方法，该方法使我们能够通过涉及去离子水洗的简单操作来重复使用细菌细胞和载体。这减轻了使用载体材料所带来的问题，并极大地有助于提高微生物生产过程的效率和生产率。
